The past two years of NIH funding have allowed us to explore the effects of ethanol (ETOH) both in vivo and in vitro on hypothalamic luteinizing hormone-releasing hormone (LHRH) release, and pituitary luteinizing hormone (LH) secretion (both basal and stimulated), microheterogeneity and bioactivity. The current proposal is an indepth extension of these studies. The effect of in vivo ETOH feeding on the synthesis of LHRH will be studied by measuring prepro-LHRH mRNA levels. In addition to synthesis, the post- translational processing of prepro-LHRH and LHRH. Next, the bioactivity of LHRH will be investigated with the use of pituitary cells from normal, non-ethanol exposed animals in culture studying LH release after the addition of LHRH obtained from the hypothalami of the ethanol-fed and control animals. The degradation of LHRH in both the hypothalamus and pituitary from ETOH-exposed and control animals will also be explored. While the above described studies investigate in vivo ethanol effects, the secretion of LHRH in vitro will be examined both basally and when stimulated by a variety of physiologically-relevant secretagogues including dopamine, norepinephrine, naloxone, prostaglandin E2, and potassium in the presence of different doses of ETOH. In addition to the hypothalamic studies, the effect of ETOH feeding on the pituitary will be further characterized. LHRH receptor number and affinity on pituitary membranes obtained from ETOH and control animals will be determined. Next, the effect of ETOH on the second messenger calcium system in pituitary cells will be examined in an attempt to quantitate changes in intracellular free calcium levels. In addition, the impact of ETOH on calmodulin translocation in the gonadotrope will be explored. Lastly, LH synthesis in the in vivo ETOH-fed animal will be examined by measuring LH mRNA. We believe that the above described studies will physiologically dissect the exact impact of ETOH on the LHRH-LH reproductive unit in an organized and concise manner. The proposed project will take approximately for years to complete.